Probing DNA polymerase-DNA interactions: examining the template strand in exonuclease complexes using 2-aminopurine fluorescence and acrylamide quenching.

The bacteriophage T4 DNA polymerase forms fluorescent complexes with DNA substrates labeled with 2-aminopurine (2AP) in the template strand; the fluorescence intensity depends on the position of 2AP. When preexonuclease complexes are formed, complexes at the crossroads between polymerase and exonuclease complexes, 2AP in the +1 position in the template strand is fully free of contacts with the adjacent bases as indicated by high fluorescence intensity and a long fluorescence lifetime of about 10.9 ns.

Evidence for rigid binding of rhodamine 6G to silica surfaces in aqueous solution based on fluorescence anisotropy decay analysis.

Strong ionic binding of the cationic probe rhodamine 6G (R6G) to the anionic surface of silica particles in water provides a convenient labeling procedure to study both particle growth kinetics and surface modification by time-resolved fluorescence anisotropy (TRFA). The decays for R6G dispersed in diluted Ludox silica sols usually fit to a sum of picosecond and nanosecond decay components, along with a significant residual anisotropy component. The origin of the nanosecond decay component (phi2) is not fully understood, and has been ascribed to wobbling of the probe on the silica surface, the presence of a subpopulation of small nanoparticles in the Ludox sol, or rapid exchange between free and bound R6G.

Two-site ionic labeling with pyranine: implications for structural dynamics studies of polymers and polypeptides by time-resolved fluorescence anisotropy.

Time-resolved fluorescence anisotropy (TRFA) is widely used to study dynamic motions of biomolecules in a variety of environments. However, depolarization due to rapid side chain motions often complicates the interpretation of anisotropy decay data and interferes with the accurate observation of segmental motions. Here, we demonstrate a new method for two-point ionic labeling of polymers and biomolecules that have appropriately spaced amino groups using the fluorescent probe 8-hydroxyl-1,3,6-trisulfonated pyrene (pyranine).

Quantifying surface coverage of colloidal silica by a cationic peptide using a combined centrifugation/time-resolved fluorescence anisotropy approach.

Recent experimental studies have shown that time-resolved fluorescence anisotropy (TRFA) is a promising methodology for in situ characterization of the surface modification of aqueous silica nanocolloids. Here we provide a more fundamental insight into the principle of this approach and discuss how the adsorption parameters for a cationic peptide, Lys-Trp-Lys (denoted using the standard shortform KWK), onto Ludox nanoparticles (NPs) are linked to the rotational dynamics of rhodamine 6G (R6G) dispersed in the KWK/Ludox mixture. First, the adsorption isotherm of KWK on hydrophilic controlled pore glass (CPG-3000) was obtained using the traditional centrifugation method, which provides the total molar amount of KWK per unit surface area of the silica.

Evaluating formation and growth mechanisms of silica particles using fluorescence anisotropy decay analysis.

At present, there is no direct experimental evidence that primary silica particles, which exist only transiently for a few seconds during the Stöber silica synthesis, can be stable in aqueous solutions. In the present work, we show that primary silica particles are formed spontaneously after the dissolution of diglycerylsilane (DGS) in aqueous solutions and remain stable for prolonged periods of time. By using time-resolved fluorescence anisotropy (TRFA), we demonstrate that this unique property of DGS is ascribed to the slow kinetics of silica particle growth in diluted sols at pH approximately 9.

Direct and indirect monitoring of peptide-silica interactions using time-resolved fluorescence anisotropy.

The present work extends the application of time-resolved fluorescence anisotropy (TRFA) of a cationic probe rhodamine 6G (R6G) in aqueous Ludox to in situ monitoring of peptide adsorption onto the silica particles. Steady-state anisotropy and TRFA of R6G in Ludox sols were measured to characterize the extent of the ionic binding of the probe to silica particles in the presence of varying levels of tripeptides of varying charge, including Lys-Trp-Lys (KWK), N-acetylated Lys-Trp-Lys (Ac-KWK), Glu-Trp-Glu (EWE), and N-acetylated Glu-Trp-Glu (Ac-EWE). The results were compared to those obtained by direct observation of peptide adsorption using the steady-state anisotropy of the intrinsic tryptophan residue.

Fluorescence anisotropy in studies of solute interactions with covalently modified colloidal silica nanoparticles.

Time-resolved anisotropy decays of a fluorescent cationic solute, rhodamine 6G (R6G), in Ludox sols were measured to characterize the extent of the ionic binding of the probe to silica particles after modification of the surface with neutral or cationic silane coupling agents. The anisotropy decays provided direct evidence for distribution of the dye between the aqueous solution (picosecond decay component) and silica particles (nanosecond decay component and residual anisotropy component, which were attributed to the wobbling motion of dye on the silica surface and to the ionically bound probe, respectively). The dye was strongly adsorbed to unmodified silica nanoparticles, to the extent that less than 1% of the dye was present in the surrounding aqueous solution.

Monitoring solute interactions with poly(ethylene oxide)-modified colloidal silica nanoparticles via fluorescence anisotropy decay.

The fluorescence-based nanosize metrology approach, proposed recently by Geddes and Birch (Geddes, C. D.; Birch, D.

Protein-doped monolithic silica columns for capillary liquid chromatography prepared by the sol-gel method: applications to frontal affinity chromatography.

The development of bioaffinity chromatography columns that are based on the entrapment of biomolecules within the pores of sol-gel-derived monolithic silica is reported. Monolithic nanoflow columns are formed by mixing the protein-compatible silica precursor diglycerylsilane with a buffered aqueous solution containing poly(ethylene oxide) (PEO, MW 10,000) and the protein of interest and then loading this mixture into a fused-silica capillary (150-250-microm i.d.

Aggregation of recombinant hepatitis B surface antigen induced in vitro by oxidative stress.

In order to examine whether oxygen radicals could be responsible for aggregation of recombinant hepatitis B surface antigen (HBsAg) during its assembly in yeast, purified HBsAg was oxidized with ammonium peroxodisulphate (AP) and analyzed by non-denaturing and denaturing size exclusion chromatography, immunoassay and immunoelectron microscopy. As a result, peroxodisulphate radicals induced a reproducible aggregation of HBsAg. At 44 mM AP, the aggregation process took a few hours and the resulting structures were large, branched and non-antigenic.

Physico-chemical characterization of recombinant hepatitis B surface antigen by a multidimensional approach.

Initially, our work was directed to respond to the question: why hepatitis B surface antigen (HBsAg) produces a very broad peak in preparative size-exclusion chromatography (SEC). For this purpose, we used a multidimensional approach based on SEC fractionation of purified HBsAg followed by the individual analysis of SEC fractions by a battery of assays, such as SEC, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme-linked immunosorbent assay and transmission electron microscopy. As a result, HBsAg particles were shown to be heterogeneous in terms of particle assembly.

Evidence for the denaturation of recombinant hepatitis B surface antigen on aluminium hydroxide gel.

Despite the complexity of the subject of protein-alum interactions, a valuable information can be obtained by analyzing the adsorbed and desorbed protein by common physico-chemical methods. In the present work, to approach the adsorption of hepatitis B surface antigen (HBsAg) on alum, the experimental data were supported by complementary analyses of the adsorbed protein by immunoelectron microscopy and the desorbed protein by denaturing size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. First, the depletion of HBsAg was investigated.

Aggregation of recombinant hepatitis B surface antigen in Pichia pastoris.

The combination of immunoaffinity and size-exclusion chromatography (SEC) is a powerful tool to analyze multiprotein particle assembly. This approach was used to investigate the source of aggregation of recombinant hepatitis B surface antigen (HBsAg) detected in purified material. As HBsAg aggregation does not originate in the stresses, such as the concentration of HBsAg solutions, temperature and chaotropic agents, it is less probable that the HBsAg aggregate is produced during the process.

Size-exclusion chromatographic study of the reduction of recombinant hepatitis B surface antigen.

The reduction of the P. pastoris-derived hepatitis B surface antigen (HBsAg) has been investigated by size exclusion chromatography performed in a detergent solution containing 0.3% sodium dodecyl sulfate (SDS) and 0.

Sodium dodecylsulfate polyacrylamide gel electrophoresis of recombinant hepatitis B surface antigen particles.

To investigate the factors leading to broadening of the recombinant hepatitis B surface antigen (HBsAg) peak in size-exclusion chromatography, the HBsAg particles eluting in different regions of the peak were subjected here to electrophoretic analysis. In nonreduced samples, the 24-kD band corresponding to the S monomer was detected when excessively large amounts of HBsAg were loaded onto the gel. Hence, some monomers are not disulfide-crosslinked in assembled particles.

Reversed-phase high-performance liquid chromatographic study of thimerosal stability in Cuban recombinant hepatitis B vaccine.

Non-degraded thimerosal was determined in the presence of its decomposition products by directly assaying recombinant hepatitis B vaccine using a reversed-phase liquid chromatographic method. Methanol-water-orthophosphoric acid (65:35:0.9, v/v/v) was used as the eluent.